The prevalence of type 2 diabetes mellitus (T2DM) and the metabolic syndrome has rapidly increased over the past several decades. Prevention and treatment of these diseases through pharmacological interventions and lifestyle changes have limited success, and novel therapeutic approaches are urgently needed. Low levels of plasma high-density lipoprotein (HDL) are a hallmark of the metabolic syndrome and an established risk factor for T2DM, and studies in humans and in animal models indicate that HDL positively influences glucose homeostasis; however, the mechanism by which HDL exerts these effects is poorly understood. Our previous work demonstrated that HDL binding to its receptor Scavenger Receptor Class B Type I (SR-BI) potently activates endothelial NO synthase (eNOS) and enhances NO production. Growing body of evidence indicates that endothelial dysfunction, particularly decreased NO production, plays a causal role in abnormal glucose and insulin homeostasis. To determine whether HDL-SR-BI in endothelial cells plays a role in glucose homeostasis, we have recently assessed metabolic phenotypes in mice with specific deletion of SR-BI in endothelial cells. We found that these mice develop glucose intolerance and insulin resistance compared with control littermates. Springboarding from these surprising discoveries, the overall goal of the proposed project is to determine the mechanism by which endothelial SR-BI regulates glucose homeostasis. Aim 1 will determine the mechanisms governing glucose homeostasis that are mediated by endothelial SR-BI. Using the mice with endothelial SR-BI deletion, Aim 1 will test the hypothesis that endothelial SR-BI plays a major role in normal glucose homeostasis by promoting skeletal muscle glucose delivery and by preventing inflammatory responses in adipose tissues. Aim 2 will identify the molecular pathway that regulates glucose homeostasis mediated by SR-BI. Roles of HDL, PDZK1 and NO will be determined by loss- and gain-of-function strategies using mouse models. We will test the hypothesis that HDL promotes insulin sensitivity through the process that requires endothelial SR-BI, PDZK1, eNOS activation and resulting production of NO. Aim 3 will determine how SR-BI uniquely initiates signaling in endothelium. Based upon our recent discovery that PDZK1 interacts with the serine/threonine kinase Bcr (Breakpoint cluster region) in endothelium, we will test the hypothesis that HDL stimulates Bcr kinase to result in phosphorylation of a Bcr substrate that links SR-BI and PDZK1 to eNOS activation and that Bcr is required for normal glucose homeostasis and insulin sensitivity afforded by HDL. By accomplishing the proposed Specific Aims, our research program will reveal novel mechanisms and thereby identify new therapeutic targets to prevent and treat insulin resistance and T2DM associated with low levels of HDL, both of which are key features of the metabolic syndrome.